The present invention relates to a process for the preparation of thermoprecipitating affinity polymers. More particularly, the present invention relates to a process for the preparation of polymers useful for the separation of enzymes of protease type exemplified by trypsin. Affinity polymers prepared by the process of the present invention exhibit stronger binding with trypsin which is useful in enhancing the recovery of trypsin from dilute aqueous solutions and from a mixture of trypsin and chymotrypsin or a mixture of trypsin and other enzymes.
Isolation and purification of biologically active macromolecules such as enzymes, from natural sources is a tedious, multi-step process, which results in very low yields and thus higher costs. As a better alternative to conventional processes, researchers have developed affinity separations based techniques for selective and enhanced separations of enzymes. The basic principle used in these techniques is to form a complex between the active site of an enzyme and inhibitor, selective and high separations are possible. Most of the affinity based operations involved polymers to which inhibitors are chemically linked. The complex formed between polymeric inhibitor and the enzyme is subsequently processed to isolate the enzyme.
Various techniques such as affinity chromatography, affinity partitioning, affinity ultrafiltration, immobilized metal affinity chromatography, affinity imprinting and affinity precipitation have been developed so far. Although all these techniques use the same basic principle of forming an enzyme-inhibitor complex, they suffer from one or the other disadvantages as follows.
Affinity chromatography uses a column containing an inhibitor or a dye or an antibody for a given enzyme for its separation from a mixture of enzymes. The solution of enzymes is poured over the affinity column to retain the desired enzyme on column for subsequent isolation. This technique is efficient only for small capacity columns. With the scale up of columns, the problems of sample pre-treatment and plugging of packed column becomes severe. [Y. Li, G. Kunyu, C. Lubai, Z. Hanfa, Z. Yunkui Sepu, 14, 415 (1996), T. Makriyannis, Y. D. Clonis, Biotech. Bioengg. 53, 49 (1997)].
In case of affinity crossflow ultrafiltration, a mixture of enzymes is filtered through a membrane containing affinity group under pressure. This technique is suitable in the cases where the difference between the molecular weights of the two enzymes is high. Also, with the increase in the filtration time, denaturation of enzymes as well as clogging of membrane takes place due to the pressure applied. [K. Sigmundsen, H. Filippusson, Polymer Int. 41, 335 (1996); T. B. Choe, P. Masse, A. Verdier, Biotech.Lett., 8, 163 (1986)].
Affinity partitioning of two-phase aqueous systems is widely used technique as compared to the methods mentioned above. In this technique, concentrated aqueous solution of poly (ethylene glycol) (PEG) with or without linking affinity group is mixed with enzyme solution containing moderate to high salt concentration. The two phases are mixed well and allowed to separate. The desired enzyme gets predominantly partitioned in one phase, which subsequently can be isolated. Disadvantages of this technique are non-specific extraction of other proteinaceous molecules along with desired enzyme and also poor interactions between enzyme and affinity group due to high ionic strength. [G. Takerkart, E. Segard, M. Monsigny, FEBS Lett., 42, 218 (1972); B. A. Andrews, D. M. Head, P. Dunthorne, J. A. Asenjo, Biotech. Tech., 4, 49 (1990)].
Immobilised metal affinity chromatography is a technique in which the columns of polymeric support containing chelated metal ions are used. These metal ions form coordination complex with histidine, tyrosine, cysteine, etc. present on the surface of the enzyme. Although this technique has advantages like high column capacity, ease in enzyme elution, etc. it is not very selective. [Ehteshami, J. Porath, R. Guzman, G. Ehteshami, J. Mol. Recognit. 9, 733 (1996); A. L. Blomkalns, M. R. Gomez, Prep. Biochem. Biotechnol. 27, 219 (1997)].
Molecular imprinting of matrices containing metal chelates is a recently developed technique, which increases the selectivity [F. H. Arnold, P. Dahl, D. Shnek, S. Plunkett, U.S. Pat. No. 5,310,648 (1994)]. In this technique complex of monomer containing chelated metal ion and enzyme is polymerised with crosslinker in order to imprint the polymer with enzyme. Although this technique exhibits a substantial selectivity, it is not as selective as that of biological antibodies or active site inhibitors of enzymes.
Compared to the techniques described above, affinity precipitation is an attractive technique from the point of view of application. [C. Senstad, B. Mattiasson, Biotech.Bioengg., 33, 216 (1989); M. Schneider, C. Guillot, B. Lamy, Ann. N.Y. Acad Sci. 369, 257 (1981); B. Mattiasson, R. Kaul, xe2x80x9cAffinity precipitationxe2x80x9d, in Molecular interactions in bioseparations, T. T. Ngo ed., Plenum Press, New xe2x80x9cYork, p 469-477 (1993); J. P. Chen, J. Ferment and Bioengg., 70, 119 (1990); I. Y. Galaev, B. Mattiasson, Biotech Bioeng. 41, 1101 (1993); M. Pecs, M. Eggert, K. Schnegerl, New Polymeric Mater. 4, 19 (1993)]. It involves formation of complex between an enzyme and a stimuli sensitive polymeric inhibitor. This complex is precipitated by pH or temperature stimulus and isolated. It is then dissociated, polymer separated by pH or temperature stimulus and the enzyme isolated. Thus, the recovery of the enzyme by this technique is much simpler and the scale up of the process is also easy. Hitherto, affinity precipitation suffers from restrictions on the accessibility of the enzyme towards the polymer bound inhibitor.
The strength of the complex formed between inhibitor and the enzyme decreases 20 to 300 fold when it is bound to the polymer. [K. B. Male, J. H. T. Luong, A. L. Nbuyen, Enzy. Microb. Tech., 9, 374 (1987); Yu, I. Galled, B. Matisson, Biotech. Bioengg. 41, 1101 (1993); J. H. T. Loung, K. B. Male, A. L. Nguyen, Biotech Bioengg., 31, 439 (1988); M. Pecs, M. Eggert, K. Schuegerl, J. Biotech., 21, 137 (1991)]. This weakening of the complex is attributed mainly to the restrictions on the free access of enzyme to the polymer bound inhibitor. The strength of the complex is expressed in terms of inhibition constant (Ki). The lower the value of KI the higher is the inhibition and stronger is the complex formed. Higher Ki values of polymeric inhibitors result in poor recovery of enzymes. Also, increased concentration of inhibitors on the high molecular weight polymers results in high KI values.
Introduction of spacers between the polymer backbone and the inhibitor is a well-known methodology used in affinity chromatography to enhance the interaction between inhibitor and the enzyme. But in affinity precipitation, the use of spacer containing polymers has not been reported so far, because the complex formation between polymer bound inhibitor and the enzyme takes place in homogeneous solution and it has been suggested that in homogeneous solutions spacers are not required.
It is therefore an object of the invention to provide a process for the preparation of thermoprecipitating affinity polymers comprising spacers between the polymer backbone and the inhibitor, useful in enhanced recovery process of trypsin by affinity precipitation.
It is another object of the invention to provide a process for the preparation of thermoprecipitating affinity polymers that exhibit enhanced interactions with the enzymes and thereby give high recovery for the desired enzymes.
Accordingly the present invention provides a process for the preparation of thermoprecipitating affinity polymers useful in the enhanced recovery of enzymes which comprises polymerising a monomer comprising a spacer and a co-monomer with a polymerisation initiator and a polymerisation accelerator at ambient temperature and pressure for a period ranging between 2 to 24 hours to obtain a polymer, linking an inhibitor to pendant carboxyl groups of the spacers in the polymer to obtain an affinity polymer by any conventional method.
In one embodiment of the invention the spacer monomer may be selected from compounds of the formula CH2xe2x95x90CRxe2x80x94COxe2x80x94NHxe2x80x94(CH2)nxe2x80x94COOH wherein R is hydrogen or methyl group and n is an integer between 1 to 10.
In another embodiment of the invention, the comonomer may be selected from compounds like N-isopropyl acrylamide, N-butyl acrylamide, N-isopropyl methacrylamide, N-vinyl caprolactam.
In a further embodiment of the invention, the molar ratio of spacer monomer to co-monomer may in the range of 1:10 to 1:1.
In another embodiment of the invention, the polymerisation initiator used may selected from compound such ammonium persulfate, potassium persulfate.
In yet another embodiment of the invention, the polymerisation initiator may be 10% to 20% by wt. of the monomers.
In yet another embodiment of the invention, the polymerisation accelerator may be selected from compounds like N,N,Nxe2x80x2,Nxe2x80x3tetramethylene ethylene diamine, sodium meta bisulfate, potassium meta bisulfate.
In another embodiment of the invention, the polymerisation accelerator is 1% to 5% by wt of the monomers.
In a further embodiment of the invention, the inhibitor may be selected from compounds like meta amino benzamidine, para amino benzamidine and their hydrochlorides.
In yet another embodiment of the invention, the molar ratio of inhibitor to carboxyl groups in the polymer is in the range of 1:1 to 10:1.
In another embodiment of the invention, the condensing reagent used for linking the inhibitor to the pendant carboxyl groups of the polymer is selected from 1-cyclohexyl 3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulphate (CMC), 1-ethyl-3-(3-Dimethylamino-propyl)carbodiimide (EDC).
In a further embodiment of the invention, the molar ratio of the condensing agent to carboxyl groups may be in the range of 1:1 to 100:1.